Compositions and methods for using platelet-rich plasma for drug discovery, cell nuclear reprogramming, proliferation or differentiation

ABSTRACT

Methods are described for using compositions containing platelet-rich plasma for the treatment of a variety of tissue lesions, and the use of compositions containing platelet-rich plasma to accelerate or complete the differentiation of a cell line. Particularly, use of platelet-rich plasma in conjunction with cell differentiation.

RELATED APPLICATIONS

This application claims the priority of Provisional Application No.61/830,754, filed Jun. 04, 2013 which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Embodiments of the invention related to platelet-rich plasma (PRP) andplatelet compositions and their use.

2. Description of the Related Art

Cell therapy is rapidly evolving in medicine. A variety of cell linesincluding embryonic stem cells (ESC), induced pluripotent stem cells(iPSCs), adult stem cells (ASCs) and progenitor cells (PCs) derived froma variety of tissues are being employed in preclinical and now clinicaltrials to treat disease. A consistent issue with the use of any type ofcell line, however, is differentiating it to an appropriate type ofcell. These pluripotent or multipotent cells need to be driven to aspecific cell line in order for them to be used for a specific diseaseor disorder.

Specifically, iPSCs can be created by overexpressing key transcriptionalfactors. Landmark work by Yamanaka and coworkers (U.S. Pat. No.8,278,104) and others have confirmed that somatic cells can betransformed into pluripotent cells via these methods. Lentiviruses,retroviruses, adenoviruses, plasmids and transposable elements have allbeen used to create these cells lines. This methodology, however, mayresult in the integration of viral DNA into the genome.

Cells lines can be differentiated into mature cell types such as amuscle cells, a skin cell or an astrocyte, a mature type of brain cell.A variety of growth factors and other cytokines can be used to pushimmature cell lines into differentiated cell types.

Unfortunately, “genetic or epigenetic errors may be introduced duringnuclear reprogramming, and the generation of tissues from pluripotentstem cells employs laboratory methods for cell generation and expansionthat increase the risk of genetic instability, epigenetic modification,and the generation of tumorigenic cells. These issues may be addressedin part by using non-viral reprogramming methods.” (Volz et al 2012)

SUMMARY OF THE INVENTION

Blood is comprised of Red Blood Cells (RBC), White Blood Cells (WBC),Plasma, and Platelets. Platelet-rich plasma (PRP) is a fractionation ofwhole blood containing concentrated platelets and white blood cells andwhich may include high quantities of cytokines such as vascularendothelial growth factor (VEGF), transforming growth factor beta(TGF-β), and platelet-derived growth factor (PDGF). Platelets areresponsible for blood clotting and when activated, release growthfactors and other bioactive molecules which are involved in stimulatingthe healing of bone and soft tissue. For example, platelets release VEGFand basic fibroblast growth factor from alpha granules and adenosinediphosphate (ADP), adenosine triphosphate (ATP), and ionized calciumfrom dense granules. White blood cells (WBCs), also known as leukocytes,are involved in defending the body against both infectious disease andforeign materials. The two most common types of white blood cells arethe lymphocytes and neutrophils. Lymphocytes secrete factors,lymphokines, which modulate the functional activities of many othertypes of cells and are often present at sites of chronic inflammation.Neutrophils, which are the most abundant white blood cell type inmammals, are recruited to the site of injury within minutes followingtrauma. Neutrophils form an essential part of the innate immune system,playing a role in inflammation.

Platelet-rich plasma (PRP) is a bioactive fraction of whole blood thathas been used to treat a variety of connective tissue disordersincluding chronic tennis elbow. Within PRP are dozens of molecules thatcould be used to help differentiate cell lines. By using an autologousfraction of whole blood to create PRP, a viral free method ofdifferentiating cells is provided. A specific example is given where adedifferentiated cell line is shown to differentiate into a specifictype of cell. The compositions and methods herein described do not limitthe invention to this cell line but it should be realized that themethods and compositions could be used in variety of cell linesincluding but not limited to ESC, iPSCs, ASCs (derived from any source)and any line of progenitor cells. Furthermore, platelet-rich plasma maybe used to create these lines by isolating the appropriate factors andthen using these factors to reprogram mature cells into pluripotentcells. These cells could then be differentiated with a different set offactors within the PRP to achieve the desired cell line. This inventionwould eliminate the need for viral derived vectors or other methods ofartificially stimulating nuclear reprograming or differentiation.Importantly, the use of PRP would enhance the yield of any reprogramingor differentiation method. Imbedded in this invention are novelcompositions and methods including specific fractions of whole bloodcontaining platelets, white blood cells, stem cells and specific growthfactors. Methods for using these compositions for small molecule or drugdiscovery are also disclosed. After using PRP to differentiate celllines, PRP may also be used in combination with these linestherapeutically. PRP may further be used in combination with agenetically engineered small molecule or drug to enhance the effects ofPRP or the small molecule. Therapeutic applications include but are notlimited to neurologic, cardiac, cardiovascular, peripheral vascular,neoplastic, musculoskeletal, metabolic, inflammatory, infectious,urologic, gynecologic, dermatologic (including cosmetic issues such aswrinkles and baldness), allergic or immunologic, ophthalmologic,obstetric, pediatric, surgical, internal medicine or orthopedicapplications. Treatment of specific problems include but are not limitedto cartilage degeneration in any joint (knee, hip, shoulder, spine),tendinopathy, bone related problems, cardiovascular injuries anddisorders such as acute or chronic heart failure, an acute heart attackand peripheral vascular disease. For clarification, the compositions andmethods in this disclosure are examples and are meant to be inclusive ofother problems and disorders.

In accordance with one aspect, a method of producing induced pluripotentstem (iPS) cells comprising obtaining fibroblast cells from skin tissuefrom an individual, administering a growth factor selected from thegroup consisting of vascular endothelial growth factor (VEGF),granulocyte colony stimulating factor (G-CSF), plerixafor, sargramostim,γ-tocotrienol, vitamin E, ancestim and bone morphogenic growth factor tothe individual, isolating platelet-rich plasma (PRP) from whole blood ofthe individual, and treating the fibroblast cells of the individual withthe isolated PRP. In some embodiments, the method wherein the wholeblood is obtained from spleen or bone marrow. In some embodiments, themethod wherein the platelet-rich plasma comprises a plateletconcentration of 151,000/microliter to 7,000,000/microliter or higher,and further comprising treating the fibroblast cells of the individualwith a bone marrow concentrate and fraction of adipose tissue. In someembodiments, the method wherein the platelet-rich plasma comprises aplatelet concentration of 151,000/microliter to 7,000,000/microliter orhigher, and further comprising CD34+ cells at a concentration 1-3×10⁹per liter to 100×10⁹ per liter or higher. In some embodiments, themethod wherein the platelet-rich plasma comprises a platelets in aconcentration of 151,000/microliter to 7,000,000/microliter or higher,and further comprising stromal-derived factor −1 (SDF-1) in aconcentration of 100 pg/ml to 5000 pg/ml. In some embodiments, themethod further comprising measuring the potency of platelet rich plasmaby visually observing it over time by at least one method selected fromthe group consisting of cellular movement, microfluidics, andchemotaxis. In some embodiments, the method wherein the PRP is used indrug discovery to identify new targets. In some embodiments, the methodwherein the PRP is used for nuclear reprogramming, cell proliferation,or differentiation. In some embodiments, the method further comprisingadministrating granulocyte colony-stimulating factor (G-CSF) or anyother molecule that stimulates the production of stem cells by the bodyprior to the preparation of platelet-rich plasma from any source.

In accordance with another aspect, a composition comprising cells froman individual, and platelet-rich plasma (PRP) isolated from whole bloodof the individual, wherein the cells of the individual are treated withthe PRP to produce induced pluripotent stem (iPS) cells. In someembodiments, the composition wherein the cells are fibroblast cells fromskin tissue from the individual. In some embodiments, the compositionfurther comprising a growth factor selected from the group consisting ofvascular endothelial growth factor (VEGF), granulocyte colonystimulating factor (G-CSF), plerixafor, sargramostim, γ-tocotrienol,vitamin E, ancestim and bone morphogenic growth factor to theindividual. In some embodiments, the composition wherein the whole bloodis obtained from spleen or bone marrow. In some embodiments, thecomposition wherein the composition comprises the platelet-rich plasmacomprising a platelets concentration of 151,000/microliter to7,000,000/microliter or higher and comprises at least one cell selectedfrom the group consisting of embryonic stem cells, induced pluripotentstem cells, adult stem cells, and progenitor cell lines. In someembodiments, the composition wherein the adult stem cells comprisemesenchymal stem cells. In some embodiments, the composition wherein thecomposition comprises a platelet-rich plasma comprising a plateletconcentration of 151,000/microliter to 7,000,000/microliter or higherand comprises progenitor or precursor stem cell lines. In someembodiments, the composition wherein the progenitor or precursor stemcell lines comprise mesenchymal stem precursor lines. In someembodiments, the composition wherein the composition comprises aplatelet-rich plasma comprising a platelet concentration of151,000/microliter to 7,000,000/microliter or higher and comprises CD34+cells at a concentration 1-3×10⁹ per liter to 100×10⁹ per liter orhigher. In some embodiments, the composition wherein the compositioncomprises a platelet-rich plasma comprising a platelets in aconcentration of 151,000/microliter to 7,000,000/microliter or higherand comprises stromal-derived factor −1 (SDF-1) in a concentration of100 pg/ml to 5000 pg/ml.

In accordance with another aspect, the composition of platelet richplasma comprising the platelet rich plasma derived from inducedpluripotent stem cells at a platelet concentration of 151,000/microliterto 7,000,000/microliter or higher and pluripotent stem cells in anyconcentration or form. In accordance with another aspect, the method ofpreparing platelet rich plasma and storing it in a reduced and orincreased oxygen concentration device.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other feature of this invention will now be described withreference to the drawings of preferred embodiments which are intended toillustrate and not to limit the invention.

FIG. 1 shows Glioblastoma cells (012705) cultured with (right panels)and without (left panels) 10% PRP for 10 days at 100× and 200×magnification.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Platelet-rich Plasma Preparation and Compositions

The term “platelet-rich plasma” or “PRP” as used herein is a broad termwhich is used in its ordinary sense and is a concentration of plateletsgreater than the peripheral blood concentration suspended in a solutionof plasma, or other excipient suitable for administration to a human ornon-human animal including, but not limited to isotonic sodium chloridesolution, physiological saline, normal saline, dextrose 5% in water,dextrose 10% in water, Ringer solution, lactated Ringer solution, Ringerlactate, Ringer lactate solution, and the like. PRP compositions may bean autologous preparation from whole blood taken from the subject to betreated or, alternatively, PRP compositions may be prepared from a wholeblood sample taken from a single donor source or from whole bloodsamples taken from multiple donor sources. In general, PRP compositionscomprise platelets at a platelet concentration that is higher than thebaseline concentration of the platelets in whole blood. In someembodiments, PRP compositions may further comprises WBCs at a WBCconcentration that is higher than the baseline concentration of the WBCsin whole blood. As used herein, baseline concentration means theconcentration of the specified cell type found in the patient's bloodwhich would be the same as the concentration of that cell type found ina blood sample from that patient without manipulation of the sample bylaboratory techniques such as cell sorting, centrifugation orfiltration. Where blood samples are obtained from more than one source,baseline concentration means the concentration found in the mixed bloodsample from which the PRP is derived without manipulation of the mixedsample by laboratory techniques such as cell sorting, centrifugation orfiltration.

In some embodiments, PRP compositions comprise elevated concentrationsof platelets and WBCs and lower levels of RBCs and hemoglobin relativeto their baseline concentrations. In some embodiments of PRPcomposition, only the concentration of platelets is elevated relative tothe baseline concentration. Some embodiments of PRP composition compriseelevated levels of platelets and WBCs compared to baselineconcentrations. In some embodiments, PRP compositions comprise elevatedconcentrations of platelets and lower levels of neutrophils relative totheir baseline concentrations. Some embodiments of PRP compositioncomprise elevated levels of platelets and neutrophil-depleted WBCscompared to their baseline concentrations. In some embodiments of PRP,the ratio of lymphocytes and monocytes to neutrophils is significantlyhigher than the ratios of their baseline concentrations.

The PRP formulation may include platelets at a level of between about1.01 and about 2 times the baseline, about 2 and about 3 times thebaseline, about 3 and about 4 times the baseline, about 4 and about 5times the baseline, about 5 and about 6 times the baseline, about 6 andabout 7 times the baseline, about 7 and about 8 times the baseline,about 8 and about 9 times the baseline, about 9 and about 10 times thebaseline, about 11 and about 12 times the baseline, about 12 and about13 times the baseline, about 13 and about 14 times the baseline, orhigher. In some embodiments, the platelet concentration may be betweenabout 4 and about 6 times the baseline. Typically, a microliter of wholeblood comprises at least 140,000 to 150,000 platelets and up to 400,000to 500,000 platelets. The PRP compositions may comprise about 500,000 toabout 7,000,000 platelets per microliter. In some instances, the PRPcompositions may comprise about 500,000 to about 700,000, about 700,000to about 900,000, about 900,000 to about 1,000,000, about 1,000,000 toabout 1,250,000, about 1,250,000 to about 1,500,000, about 1,500,000 toabout 2,500,000, about 2,500,000 to about 5,000,000, or about 5,000,000to about 7,000,000 platelets per microliter.

The WBC concentration is typically elevated in PRP compositions. Forexample, the WBC concentration may be between about 1.01 and about 2times the baseline, about 2 and about 3 times the baseline, about 3 andabout 4 times the baseline, about 4 and about 5 times the baseline,about 5 and about 6 times the baseline, about 6 and about 7 times thebaseline, about 7 and about 8 times the baseline, about 8 and about 9times the baseline, about 9 and about 10 times the baseline, or higher.The WBC count in a microliter of whole blood is typically at least 4,100to 4,500 and up to 10,900 to 11,000. The WBC count in a microliter ofthe PRP composition may be between about 8,000 and about 10,000; about10,000 and about 15,000; about 15,000 and about 20,000; about 20,000 andabout 30,000; about 30,000 and about 50,000; about 50,000 and about75,000; and about 75,000 and about 100,000.

Among the WBCs in the PRP composition, the concentrations may vary bythe cell type but, generally, each may be elevated. In some variations,the PRP composition may comprise specific concentrations of varioustypes of white blood cells. The relative concentrations of one cell typeto another cell type in a PRP composition may be the same or differentthan the relative concentration of the cell types in whole blood. Forexample, the concentrations of lymphocytes and/or monocytes may bebetween about 1.1 and about 2 times baseline, about 2 and about 4 timesbaseline, about 4 and about 6 times baseline, about 6 and about 8 timesbaseline, or higher. In some variations, the concentrations of thelymphocytes and/or the monocytes may be less than the baselineconcentration. The concentrations of eosinophils in the PRP compositionmay be less than baseline, about 1.5 times baseline, about 2 timesbaseline, about 3 times baseline, about 5 times baseline, or higher.

In whole blood, the lymphocyte count is typically between 1,300 and4,000 cells per microliter, but in other examples, the lymphocyteconcentration may be between about 5,000 and about 20,000 permicroliter. In some instances, the lymphocyte concentration may be lessthan 5,000 per microliter or greater than 20,000 per microliter. Themonocyte count in a microliter of whole blood is typically between 200and 800. In the PRP composition, the monocyte concentration may be lessthan about 1,000 per microliter, between about 1,000 and about 5,000 permicroliter, or greater than about 5,000 per microliter. The eosinophilconcentration may be between about 200 and about 1,000 per microliterelevated from about 40 to 400 in whole blood. In some variations, theeosinophil concentration may be less than about 200 per microliter orgreater than about 1,000 per microliter.

In certain variations, the PRP composition may contain a specificconcentration of neutrophils. The neutrophil concentration may varybetween less than the baseline concentration of neutrophils to eighttimes than the baseline concentration of neutrophils. In someembodiments, the PRP composition may include neutrophils at aconcentration of 50-70%, 30-50%, 10-30%, 5-10%, 1-5%, 0.5-1%, 0.1-0.5%of levels of neutrophils found in whole blood or even less. In someembodiments, neutrophil levels are depleted to 1% or less than thatfound in whole blood. In some variations, the neutrophil concentrationmay be between about 0.01 and about 0.1 times baseline, about 0.1 andabout 0.5 times baseline, about 0.5 and 1.0 times baseline, about 1.0and about 2 times baseline, about 2 and about 4 times baseline, about 4and about 6 times baseline, about 6 and about 8 times baseline, orhigher. The neutrophil concentration may additionally or alternativelybe specified relative to the concentration of the lymphocytes and/or themonocytes. One microliter of whole blood typically comprises 2,000 to7,500 neutrophils. In some variations, the PRP composition may compriseneutrophils at a concentration of less than about 1,000 per microliter,about 1,000 to about 5,000 per microliter, about 5,000 to about 20,000per microliter, about 20,000 to about 40,000 per microliter, or about40,000 to about 60,000 per microliter. In some embodiments, neutrophilsare eliminated or substantially eliminated. Means to deplete bloodproducts, such as PRP, of neutrophils is known and discussed in U.S.Pat. No. 7,462,268, which is incorporated herein by reference.

Several embodiments are directed to PRP compositions in which levels ofplatelets and white blood cells are elevated compared to whole blood andin which the ratio of monocytes and/or lymphocytes to neutrophils ishigher than in whole blood. The ratio of monocytes and/or lymphocytes toneutrophils may serve as an index to determine if a PRP formulation maybe efficaciously used as a treatment for a particular disease orcondition. PRP compositions where the ratio of monocytes and/orlymphocytes to neutrophils is increased may be generated by eitherlowering neutrophils levels, or by maintaining neutrophil levels whileincreasing levels of monocytes and/or lymphocytes. Several embodimentsrelate to a PRP formulation that contains 1.01 times, or higher,baseline platelets in combination with a 1.01 times, or higher, baselinewhite blood cells with the neutrophil component depleted at least 1%from baseline.

In some embodiments, the PRP compositions may comprise a lowerconcentration of red blood cells (RBCs) and/or hemoglobin than theconcentration in whole blood. The RBC concentration may be between about0.01 and about 0.1 times baseline, about 0.1 and about 0.25 timesbaseline, about 0.25 and about 0.5 times baseline, or about 0.5 andabout 0.9 times baseline. The hemoglobin concentration may be depressedand in some variations may be about 1 g/dl or less, between about 1 g/dland about 5 g/dl, about 5 g/dl and about 10 g/dl, about 10 g/dl andabout 15 g/dl, or about 15 g/dl and about 20 g/dl. Typically, wholeblood drawn from a male patient may have an RBC count of at least4,300,000 to 4,500,000 and up to 5,900,000 to 6,200,000 per microliterwhile whole blood from a female patient may have an RBC count of atleast 3,500,000 to 3,800,000 and up to 5,500,000 to 5,800,000 permicroliter. These RBC counts generally correspond to hemoglobin levelsof at least 132 g/L to 135 g/L and up to 162 g/L to 175 g/L for men andat least 115 g/L to 120 g/L and up to 152 g/L to 160 g/L for women.

In some embodiments, PRP compositions contain increased concentrationsof growth factors and other cytokines. In several embodiments, PRPcompositions may include increased concentrations of one or more of:platelet-derived growth factor, transforming growth factor beta,fibroblast growth factor, insulin-like growth factor, insulin-likegrowth factor 2, vascular endothelial growth factor, epidermal growthfactor, interleukin-8, keratinocyte growth factor, and connective tissuegrowth factor. In some embodiments, the platelets collected in PRP areactivated by thrombin and calcium chloride to induce the release ofthese growth factors from alpha granules.

In some embodiments, a PRP composition is activated exogenously withthrombin and/or calcium to produce a gel that can be applied to an areato be treated. The process of exogenous activation, however, results inimmediate release of growth factors. Other embodiments relate toactivation of PRP via in vivo contact with collagen containing tissue atthe treatment site. The in vivo activation of PRP results in slowergrowth factor release at the desired site.

Methods of Making

The PRP composition may comprise a PRP derived from a human or animalsource of whole blood. The PRP may be prepared from an autologoussource, an allogenic source, a single source, or a pooled source ofplatelets and/or plasma. To derive the PRP, whole blood may becollected, for example, using a blood collection syringe. The amount ofblood collected may depend on a number of factors, including, forexample, the amount of PRP desired, the health of the patient, theseverity or location of the tissue damage and/or the MI, theavailability of prepared PRP, or any suitable combination of factors.Any suitable amount of blood may be collected. For example, about 1 ccto about 150 cc of blood or more may be drawn. More specifically, about27 cc to about 110 cc or about 27 cc to about 55 cc of blood may bewithdrawn. In some embodiments, the blood may be collected from apatient who may be presently suffering, or who has previously sufferedfrom, connective tissue damage and/or an MI. PRP made from a patient'sown blood may significantly reduce the risk of adverse reactions orinfection.

In an exemplary embodiment, about 55 cc of blood may be withdrawn into a60 cc syringe (or another suitable syringe) that contains about 5 cc ofan anticoagulant, such as a citrate dextrose solution. The syringe maybe attached to an apheresis needle, and primed with the anticoagulant.Blood (about 27 cc to about 55 cc) may be drawn from the patient usingstandard aseptic practice. In some embodiments, a local anesthetic suchas anbesol, benzocaine, lidocaine, procaine, bupivicaine, or anyappropriate anesthetic known in the art may be used to anesthetize theinsertion area.

The PRP may be prepared in any suitable way. For example, the PRP may beprepared from whole blood using a centrifuge. The whole blood may or maynot be cooled after being collected. Isolation of platelets from wholeblood depends upon the density difference between platelets and redblood cells. The platelets and white blood cells are concentrated in thelayer (i.e., the “buffy coat”) between the platelet depleted plasma (toplayer) and red blood cells (bottom layer). For example, a bottom buoyand a top buoy may be used to trap the platelet-rich layer between theupper and lower phase. This platelet-rich layer may then be withdrawnusing a syringe or pipette. Generally, at least 60% or at least 80% ofthe available platelets within the blood sample can be captured. Theseplatelets may be resuspended in a volume that may be about 3% to about20% or about 5% to about 10% of the sample volume.

In some examples, the blood may then be centrifuged using agravitational platelet system, such as the Cell Factor Technologies GPSSystem® centrifuge. The blood-filled syringe containing between about 20cc to about 150 cc of blood (e.g., about 55 cc of blood) and about 5 cccitrate dextrose may be slowly transferred to a disposable separationtube which may be loaded into a port on the GPS centrifuge. The samplemay be capped and placed into the centrifuge. The centrifuge may becounterbalanced with about 60 cc sterile saline, placed into theopposite side of the centrifuge. Alternatively, if two samples areprepared, two GPS disposable tubes may be filled with equal amounts ofblood and citrate dextrose. The samples may then be spun to separateplatelets from blood and plasma. The samples may be spun at about 2000rpm to about 5000 rpm for about 5 minutes to about 30 minutes. Forexample, centrifugation may be performed at 3200 rpm for extraction froma side of the separation tube and then isolated platelets may besuspended in about 3 cc to about 5 cc of plasma by agitation. The PRPmay then be extracted from a side port using, for example, a 10 ccsyringe. If about 55 cc of blood may be collected from a patient, about5 cc of PRP may be obtained.

As the PRP composition comprises activated platelets, active agentswithin the platelets are released. These agents include, but are notlimited to, cytokines (e.g., IL-1B, IL-6, TNF-A), chemokines (e.g.,ENA-78 (CXCL5), IL-8 (CXCL8), MCP-3 (CCL7), MIP-1A (CCL3), NAP-2(CXCL7), PF4 (CXCL4), RANTES (CCL5)), inflammatory mediators (e.g.,PGE2), and growth factors (e.g., Angiopoitin-1, bFGF, EGF, FGF, HGF,IGF-I, IGF-II, PDAF, PDEGF, PDGF AA and BB, TGF-.beta. 1, 2, and 3, andVEGF).

The PRP composition may be delivered as a liquid, a solid, a semi-solid(e.g., a gel), an inhalable powder, or some combination thereof. Whenthe PRP is delivered as a liquid, it may comprise a solution, anemulsion, a suspension, etc. A PRP semi-solid or gel may be prepared byadding a clotting agent (e.g., thrombin, epinephrine, calcium salts) tothe PRP. The gel may be more viscous than a solution and therefore maybetter preserve its position once it is delivered to target tissue. Insome embodiments, the delivery to the target tissue can include deliveryto a treatment area in the body as well as incorporation into cellcultures or suspensions as described herein. In some embodiments, thePRP composition is delivered without a clotting agent.

In some instances, it may be desirable to deliver the PRP composition asa liquid and have it gel or harden in situ. For example, the PRPcompositions may include, for example, collagen, cyanoacrylate,adhesives that cure upon injection into tissue, liquids that solidify orgel after injection into tissue, suture material, agar, gelatin,light-activated dental composite, other dental composites, silk-elastinpolymers, Matrigel® gelatinous protein mixture (BD Biosciences),hydrogels and/or other suitable biopolymers. Alternatively, the abovementioned agents need not form part of the PRP mixture. For example, theabove mentioned agents may be delivered to the target tissue before orafter the PRP has been delivered to the target tissue to cause the PRPto gel. In some embodiments, the PRP composition may harden or gel inresponse to one or more environmental or chemical factors such astemperature, pH, proteins, etc.

The PRP may be buffered using an alkaline buffering agent to aphysiological pH. The buffering agent may be a biocompatible buffer suchas HEPES, TRIS, monobasic phosphate, monobasic bicarbonate, or anysuitable combination thereof that may be capable of adjusting the PRP tophysiological pH between about 6.5 and about 8.0. In certainembodiments, the physiological pH may be from about 7.3 to about 7.5,and may be about 7.4. For example, the buffering agent may be an 8.4%sodium bicarbonate solution. In these embodiments, for each cc of PRPisolated from whole blood, 0.05 cc of 8.4% sodium bicarbonate may beadded. In some embodiments, the syringe may be gently shaken to mix thePRP and bicarbonate.

As noted above, the PRP composition may comprise one or more additionalagents, diluents, solvents, or other ingredients. Examples of theadditional agents include, but are not limited to, thrombin,epinephrine, collagen, calcium salts, pH adjusting agents, materials topromote degranulation or preserve platelets, additional growth factorsor growth factor inhibitors, NSAIDS, steroids, anti-infective agents,and mixtures and combinations of the foregoing.

In some embodiments, the PRP compositions may comprise a contrast agentfor detection by an imaging technique such as X-rays, magnetic resonanceimaging (MRI), or ultrasound. Examples of such contrast agents include,but are not limited to, X-ray contrast (e.g., IsoVue), MRI contrast(e.g., gadolinium), and ultrasound contrast.

The PRP composition may be modified using a filtration device and/orcell sorter. The filtration device may use vacuum and/or gravity toremove a portion of the platelet, WBCs, and/or RBCs. In some variations,a cell sorter may receive a CBC input from an automated blood analyzerand/or a gene chip reader. A user may select or confirm one or moremodifications to be made to the PRP composition. Of course, the cellsorter may be used with whole blood, portions of whole blood, and/orPRP. The cell sorter may sort the PRP composition based on electriccharge, density, size, deformation, fluorescence, or the like. Examplesof cell sorters include the BD FACSAria® cell sorter, the CytopeiaInFlux® cell sorter, those manufactured by Beckman Coulter, the CytonomeGigasort® cell sorter, and the like.

PRP Compositions According to Embodiments of the Invention

PRP is prepared by any method including but not limited to cell sorting,centrifugation, gravity filtration or other methods. The compositiondescribed herein can be created by a cell sorter device that useselectric charge, density, size, deformation, fluorescence or other meansto achieve the composition. As part of this invention a variety ofmethods and compositions are claimed as novel.

Granulocyte-colony stimulating factor (GCSF) can mobilize cells frombone marrow including platelets. Pretreating patients with GCSF willenhance the value of PRP by increasing the number of platelets and otherreparative cells. This may be especially true in patients with lowplatelet counts or in older patients that have lower cell counts. GCSFor other molecules that mobilize cells are given once, twice or manytimes prior to drawing whole blood from any source as described above. Apreferred protocol would be to give GCSF for several days prior to thePRP isolation.

One specific method is to administer granulocyte colony-stimulatingfactor (G-CSF, also known as pegfilgrastim) or any other molecule thatstimulates the production of stem cells by the body to a patient priorto the preparation of PRP. 5-50 micrograms per kilogram per day isadministered to a patient for 1-10 days. PRP is then prepared from wholeblood of the patient. The whole blood may be from any source includingbone marrow and spleen. In a preferred embodiment, 10 micrograms perkilogram per day of G-CSF is administered subcutaneously to a patientfor 1-10 days. PRP is then prepared from whole blood of the patient fromany source including bone marrow or the spleen. Additional medicationsor newer molecules that stimulate blood or stem cell production could beadded or substituted. Embodiments of the invention are directed to theuse of a molecule to stimulate endogenous production of desired cellsprior to preparing the PRP or bone marrow concentrate to be usedtherapeutically or for the reprogramming, proliferation, ordifferentiation of cells. Examples of other potential molecules includebut are not limited to: plerixafor, sargramostim, gamma-tocotrienol,vitamin E and ancestim. As science evolves, newer molecules that helpmobilize desired cells will be developed. It is anticipated by thisfiling that these newer molecules would be included in the list thatcould be administered prior to the production of PRP or other bioactiveblood fractions.

By giving a patient such a mobilizing drug prior to preparing PRP, itshould be noted that the composition will change compared to withoutsuch a cell mobilization drug. Therefore, a variety of compositions areclaimed to be novel and useful.

In alternate embodiments, the molecule, such as G-CSF is administered atthe same time that the PRP is administered or the molecule, such asG-CSF, is administered after the administration of PRP for a period tobe determined depending on the treatment. For example, for treatment ofa connective tissue injury, G-CSF with or without PRP may beadministered over a period of weeks of months on a regular basis such asonce a week, twice a week, three times per week or more as determined bythe medical practitioner. In some embodiments, the molecule, such asG-CSF is administered before blood is taken from the patient forisolation of PRP for a period of 1-10 days as well as at the same timeas administration of PRP to the patient and/or after administration ofPRP as described above. PRP contains cytokines such as SDF-1 that arechemoattractive for stem cells. In some embodiments, the use of GCSFwould enhance the value of the PRP treatment by mobilizing more cells.

In some embodiments, the PRP could also be used in combination withother drugs or fillers such as calcium phosphate, collagen or even withbotox.

Embodiments of the invention are directed to mobilizing cells from bonemarrow using GCSF or other similar type molecules prior to theproduction of PRP of any type.

Methods of Use

Embodiments of the invention are directed to the use of that PRP for avariety of research and clinically applications including but notlimited to:

-   Cell culture of any type.-   Connective Tissue Disorders of any type including but not limited    to: tennis elbow, carpal tunnel syndrome, deQuervain's    tenosynovitis, medial epicondylitis, ulnar collateral ligament    injuries of the elbow, cartilage injuries of any type, cartilage    degeneration of any type or location, rotator cuff disease including    partial and complete tears, labral tears, biceps tendon disorders,    fractures of any type, cervical radiculopathy, acute or chronic    spinal cord injuries, disc herniations, disc degeneration, spinal    disorders of any type including spinal stenosis and facet    arthropathy, acute bony injuries including subchondral edema,    tendinopathy of any type including gluteal, quadriceps, patellar,    achilles and others, osteoarthritis of any joint, rheumatoid    arthritis, arthritis of any type, achilles tendon tears, muscle    injuries of any type or severity, or any other musculoskeletal    injury or disorder.-   Cardiovascular disorders of any type including but not limited to:    acute myocardial infarction, acute or chronic heart failure and    peripheral vascular disease of any grade.-   Neurologic injuries or disorders of any type including Parkinson's    disease, brain cancer or Alzheimer's disease.-   Wound healing. The methods outlined above could be used to    specifically treat acute or chronic wounds.-   Infections of any type.-   Dermatologic or Cosmetic Injuries or disorders such baldness,    wrinkles, burns or even skin cancer.-   Internal organ injuries or disorders such as diabetes, hernias or    fistulas.

It should be recognized that the concept is to give a cell mobilizationagent either before, during, and/ or after preparing and using PRP as atreatment for any medical issue in humans or animals.

The PRP composition may be delivered at any suitable dose. In someembodiments, the dose may be between about 1 cc and about 3 cc, betweenabout 3 cc and about 5 cc, between about 5 cc and about 10 cc, betweenabout 10 cc and about 20 cc, or more. The dose may be deliveredaccording to a medical procedure (e.g., at specific points in aprocedure) and/or according to a schedule. For example, prior to anelective cardioversion, the PRP composition may be delivered about 24hours, about 12 hours, about 6 hours, about 2 hours, and/ or about 1hour before the procedure begins.

In some embodiments, the PRP composition may be delivered to tissuedamaged by ischemia or reperfusion injury. The list of tissues includes,but is not limited to, the heart, ischemic limbs, ischemic or damagedorgans including the brain and skin. The

PRP composition may be delivered to an individual in need thereof byinjection using a syringe or catheter. The PRP composition may also bedelivered via delivery device such as a dermal patch, a spray device,sutures, stents, screws, plates, or some other implantable medicaldevice such as bioresorbable tissue patch. The PRP composition may beused as a coating or incorporated into the delivery device. The PRPdelivery device may be incubated with PRP prior to use. Incubation timesmay be from a few seconds up to any convenient time such as a fewseconds to hours before use, such as less than 1 minute, 5-10 minutes,10 minutes to an hour, 1-3 hours, 4-12 hours, 13-24 hours, 1-3 days, or3-31 days. PRP compositions may be used in conjunction with an ointment,bone graft, or drug.

The PRP alone or in combination with a delivery device may beconveniently stored in an appropriate chamber. In some embodiments, thePRP and/or PRP combined delivery device may be stored frozen and/orunder reduced oxygen concentration or increased oxygen concentration,low and/or high pH, low and/or high pressure, low and/or high UV orother light conditions, low and/or high temperature. Storage times mayvary from such as less than 1 minute, 5-10 minutes, 10 minutes to anhour, 1-3 hours, 4-12 hours, 13-24 hours, 1-3 days, 3-31 days, or 1-12months or 1-5 years. The PRP composition alone or in combination withthe delivery device may then be used clinically as appropriate.

In one exemplary embodiment, a platelet rich plasma composition isprepared and combined with a stent in an appropriate low oxygen chamberfor 1-30 minutes, preferably about 10 minutes. The chamber is thenexposed to ultraviolet light for a brief period of time, such as 1-60seconds, 1-5 minutes, or 5-15 minutes. The stent is then removed fromthe chamber and implanted into a patient. It is expected that thischamber will improve the biologic activity of the platelet rich plasmaand or device.

The site of delivery of the PRP composition is typically at or near thesite of tissue damage. The site of tissue damage may be determined bywell-established methods including imaging studies and patient feedbackor a combination thereof. The preferred imaging method used may bedetermined based on the tissue type. Commonly used imaging methodsinclude, but are not limited to MRI, X-ray, CT scan, Positron Emissiontomography (PET), Single Photon Emission Computed Tomography (SPECT),Electrical Impedance Tomography (EIT), Electrical Source Imaging (ESI),Magnetic Source Imaging (MSI), laser optical imaging NOGA mapping andultrasound techniques. The patient may also assist in locating the siteof tissue injury or damage by pointing out areas of particular painand/or discomfort.

The PRP compositions described herein may also be used to treatperipheral vascular disease, strokes or other ischemic areas such as akidney that was damaged. PRP compositions could also be used as aprimary or secondary treatment for pulmonary disease.

In some examples, a PRP composition may be used to treat a patientdiagnosed with an acute myocardial infarction or ischemic heart disease.Treatment with the PRP composition may occur in the field or in theemergency room setting. Criteria for PRP composition treatment mayinclude positive cardiac markers, ST-elevations, or new wall motionabnormalities identified on echocardiogram, for example. The decision totreat with a PRP composition, and the treatment location(s), may dependupon one or more characteristics of the myocardial infarction. Forexample, a myocardial infarction may be characterized as a ST-elevationmyocardial infarction (STEMI) or non-ST-elevation myocardial infarction(NSTEMI), a Q-wave or non-Q-wave myocardial infarction, and whether theyare subendocardial or transmural. Myocardial infarctions may also becharacterized anatomically by cardiac wall region and/or the suspectedblockage site in the cardiac vasculature. Myocardial infarctions mayalso be characterized as anterior, lateral, inferior, posterior, septal,or right-ventricular in location, and may involve disease or blockage ofthe left-anterior descending, left circumflex, left main,posterior-descending and right coronary arteries, for example.

In some embodiments, timing of the PRP preparation and application maybe based upon other treatments that are indicated in a patient with amyocardial infarction or ischemic heart disease. In some embodiments, aPRP composition may be prepared and delivered before, during, and/orafter reperfusion therapy is performed to treat an acute myocardialinfarction, a previous myocardial infarction, or ischemic heart disease.Reperfusion therapies may include thrombolytic therapy (such as heparin,TPA and or other pharmacologic agents), angioplasty, stenting (includingbare metal stents and drug-eluting stents) or coronary artery bypassgraft (CABG) surgery. In some instances, reperfusion therapy may beassociated with an increased risk of an arrhythmia, including suddendeath. Also, it is believed that the etiology of reperfusion arrhythmiasor reperfusion arrhythmia risk may be different from the arrhythmiaetiologies associated with the myocardial infarction itself For example,some reperfusion arrhythmias may be caused by triggered activity and/orre-entry. In some embodiments, PRP composition is prepared before or atthe start of a reperfusion procedure, but not used unless an arrhythmiaoccurs during the procedure. In other embodiments, the patient may beprophylactically pre-treated with a PRP composition before reperfusionoccurs, e.g., before guidewire passage across an occlusion, stentpositioning, stent expansion, or reestablishment of coronary flowthrough a bypass segment.

In some embodiments, the PRP composition is injected into or near aninfarct site. The location of the infarct site may be determined orapproximated using various techniques. For example, in some variations,diagnostic procedures such as an electrophysiology study or anelectrical mapping study of the heart may be used. In other variations,one or more imaging technologies such as MRI, X-ray, CT scan, PositronEmission tomography (PET), Single Photon Emission Computed Tomography(SPECT), Electrical Impedance Tomography (EIT), Electrical SourceImaging (ESI), Magnetic Source Imaging (MSI), NOGA mapping, laseroptical imaging and ultrasound techniques may be used. Othertechnologies and approaches that may be used include visual inspectionduring open chest surgical procedures, localized blood flowdeterminations, local electrical and structural activity, nuclearcardiology, echocardiography, echocardiographic stress test, coronaryangiography, magnetic resonance imaging (MRI), computerized tomography(CT) scans, and ventriculography.

PRP compositions that are formulated as gels or other viscous fluids maybe difficult to deliver via a needle or syringe. Thus, in variationswhere the use of a needle or syringe is desirable, a gelling and/orhardening agent may be optionally added to the PRP composition in situ.One or more needles or catheters may be configured to deliver the PRPcomposition and/or the gelling or hardening agent simultaneously, orsubstantially simultaneously, to the cardiac tissue. For example, if aneedle is used to deliver the PRP composition, the needle may comprise aplurality of lumens through which the PRP composition and the agentseparately travel. Alternatively or additionally, separate needles maybe used to deliver the components to the tissue at the same time or oneafter the other.

The PRP composition may be delivered minimally invasively and/orsurgically. For example, the PRP composition may be delivered to theheart using a catheter inserted into the patient via the femoral vein orartery, the internal jugular vein or artery, or any other suitable veinor artery. The PRP composition may be delivered along with one or moremedical devices, instruments, or agents to treat the MI and/or othercardiac conditions.

To deliver a PRP composition to the ischemic tissue, a physician may useone of a variety of access techniques. These include surgical (e.g.,sternotomy, thoracotomy, mini-thoracotomy, sub-xiphoidal) approaches,endoscopic approaches (e.g., intercostal and transxiphoidal) andpercutaneous (e.g., transvascular, endocardial, and pericardial)approaches. Once access has been obtained, the composition may bedelivered via epicardial, endocardial, or transvascular approaches. Thecomposition may be delivered to the cardiac wall tissue or cardiacvessels in one or more locations. This includes intra-myocardial,sub-endocardial, and/or sub-epicardial administration.

Upon gaining access to the ischemic tissues of the heart, the deliverydevice may be inserted through any appropriate vessel. The distal end ofthe delivery device may be then placed against the surface of themyocardium and one or more needles may be advanced into tissue.Following delivery of one or more components of the PRP composition, theneedles, if any, may be retracted. Mapping or guidance systems that relyupon voltage, ultrasound or pressure in addition to other systems may beused in combination with injection. The delivery device may then berepositioned for additional delivery of one or more components of thecomposition or may be removed from the patient. Incisions may then beclosed using standard techniques.

The delivery system may deliver the components of the PRP composition ina prescribed ratio (e.g., a ratio of the lymphocytes and theneutrophils). The prescribed ratio may be calculated beforehand ordetermined on an ad hoc basis once delivery begins. To deliver thecomponents in the prescribed ratio, the delivery device may include oneor more gears having a corresponding gear ratio, one or more lumenshaving a proportional lumen size, or any other suitable mechanism. Somedelivery devices may include one or more mixing chambers. The multiplecomponents may be delivered using separate delivery devices or may bedelivered one after the other using the same delivery device.

The delivery devices may be advanced through a vessel adjacent to theischemic tissue to be treated. The PRP composition may be injecteddirectly into the ischemic tissue using a needle and/or a needle-tipcatheter. The PRP composition may alternatively or additionally beinfused into the vessel.

When the PRP compositions are delivered using one or more catheters, anysuitable catheter may be used. For example, the catheters may includeone or more lumens and staggered or flush tips. The catheters mayinclude needles or other devices (e.g., imaging devices) located at thedistal end, and plungers or any other control located at the proximalend. The catheters and/or other delivery devices may have differentlysized lumens to deliver multiple components of the PRP composition inthe prescribed ratio. When catheters are used, a physician may navigateto the heart using one of the routes known for accessing the heartthrough the vasculature, including but not limited to navigation to aheart chamber for epicardial, endocardial, and/or transvascular deliveryof the PRP composition.

Endocardial delivery of the PRP composition may comprise accessing atreatment site, for example, in the left ventricle of a heart, using adelivery device advanced percutaneously in an anterograde approachthrough the superior vena cava or inferior vena cava into the rightventricle. The delivery device may be passed through the interatrialseptum into the left atrium and then into the left ventricle to reachtreatment site. Alternatively, the device may be advanced using atransseptal procedure, e.g., through the intraventricular septum intothe left ventricle. In another embodiment, the PRP composition may beinjected directly into the intraventricular septum from the rightventricle. An alternative endocardial delivery method may compriseaccessing the treatment site using a delivery device advancedpercutaneously in a retrograde approach through the aorta into the leftatrium and then into the left ventricle.

Transvascular delivery of compositions may comprise passing the deliverydevice through the coronary sinus into the cardiac venous system via thecardiac veins and, if needed, leaving the veins by tracking throughmyocardial tissue. An alternative transvascular delivery methodcomprises accessing a treatment site through the aorta into a coronaryartery to reach treatment site.

A practitioner may make multiple deliveries into various locations usinga single device, make multiple deliveries into various locations usingmultiple devices, make a single delivery to a single location using asingle device, or make a single delivery to a single location usingmultiple devices. The delivery devices may include at least one reusableneedle or catheter. Some embodiments may include delivery devices havingan automated dosing system (e.g., a syringe advancing system). Theautomated dosing system may allow each dose to be pre-determined anddialed in (may be variable or fixed). In some embodiments, aniontophoresis device may be used to deliver the PRP composition into theischemic tissue.

It may be desirable to deliver the PRP composition to the ischemictissues while avoiding coincidental delivery to other cardiac tissues orother locations adjacent to the heart. For example, the PRP compositionmay gel or harden upon delivery to prevent migration. In otherembodiments, the PRP compositions may be delivered without a gellingagent/activator such as thrombin. In some variations, a balloon cathetermay be placed in the coronary sinus and inflated during delivery untilthe PRP composition has solidified or at least partially immobilized.Other variations may include a pressure control system on the deliverydevice to prevent pressure-driven migration of the PRP composition.Backbleed may also be prevented by keeping the needle in place forseveral seconds (e.g., about 5 to about 30 seconds, or about 5 to about120 seconds) following an injection.

Sensors may be used to direct the delivery device to a desired locationand/or to deliver the PRP composition. For example, real-time recordingof electrical activity (e.g., an ECG), pH, oxygenation, metabolites suchas lactic acid, CO2, or the like may be used. The sensors may be one ormore electrical sensors, fiber optic sensors, chemical sensors, imagingsensors, structural sensors, and/or proximity sensors that measureconductance. The sensors may be incorporated into the delivery device orbe separate from the delivery device. In some embodiments, the sensorsmay sense and/or monitor such things as needle insertion depth, bloodgas, blood pressure or flow, hemocrit, light, temperature, vibration,voltage, electric current, power, and/or impedance. The sensors mayinclude one or more imaging systems and may be coupled to anyappropriate output device, for example, a LCD or CRT monitor whichreceives and displays information.

The total volume of the PRP composition delivered to the patient may bebased on the size of the heart, the amount of the affected ischemictissue, and/or the desired outcome of the procedure. For example, thetotal volume of composition injected may be less than 15000 μL.

The number of delivery sites in the heart may be based on the type andlocation of the infarct(s), the desired location of the PRP composition,and the distance separating the desired locations. The number ofdelivery sites may range from about 1 to about 25 sites. The distanceseparating delivery sites may vary based on the desired volume of PRP tobe delivered per delivery site, the desired total volume to bedelivered, and/or the condition of the ischemic tissue. At the deliverysite, the PRP composition may be injected, infused, or otherwisedisposed at or adjacent to the ischemic tissue. The PRP composition mayalso be infused into the vasculature (i.e., vessels) upstream of thetarget site, so that it will flow towards the affected ischemic tissue.

The location of the delivery sites may vary based on the size and shapeof the ischemic tissue, and the desired extent of the treatment of thetissue. For example, the PRP composition may be delivered into theischemic tissue, and/or into the tissue that bordering the ischemictissue. Similarly, the composition may be delivered to any combinationof the regions of ischemic tissue and other cardiac tissue.

The timing of PRP delivery relative to an acute MI may be based on theseverity of the infarction, the extent of the ischemic tissue, thecondition of the patient, and the progression of any concurrent MI orarrhythmia treatments. The PRP composition may be delivered at anysuitable time. For example, it may be delivered immediately after theonset of an MI, within one hour of an MI, one to eight hours followingan MI, or three to four days after an MI after clinical stabilization ofthe patient when it is safer for the patient to undergo a separateprocedure. Treatment may also be done later. The timing may be basedupon the level of caspase-3 in the blood. In some variations, thecomposition is delivered about one week, about 1 to about 3 weeks, about1 to about 6 months, or even up to or more than about 1 year after theMI. Treatment may be done for patients with congestive heart failure,cardiomyopathy or other heart disorders. Other times for injectingcompositions into the ischemic tissue are also contemplated, includingprior to any potential MI, and immediately upon finding an area ofischemic tissue. Of course, compositions may be injected into theischemic tissue years after an MI.

As mentioned previously, a PRP composition may additionally oralternatively be used in other cardiac procedures. These cardiacprocedures may include anti-arrhythmia procedures, procedures to correctcongenital heart defects, or other pathologies. Examples of othercardiac procedures include, but are not limited to, angioplasty,coronary artery bypass, Minimally Invasive Direct Coronary Artery Bypass(MIDCAB), off-pump coronary artery bypass, Totally Endoscopic CoronaryArtery Bypass (TECAB), aortic valve repair, aortic valve replacement,mitral valve repair, mitral valve replacement, Ross procedure, Bentallprocedure, pulmonary thromboendarterectomy, transmyocardialrevascularization (TMR), valve-sparing aortic root replacement,cardiomyoplasty, Dor procedure, heart transplantation, septal myectomy,ventricular reduction, pericardiocentesis, pericardiectomy, atrialseptostomy, Blalock-Taussig shunt procedure, Fontan procedure, Norwoodprocedure, Rastelli procedure, Maze procedure (Cox maze and minimaze),and/or pacemaker insertion. The PRP composition may be used to preventan arrhythmia associated with reperfusion of the cardiac tissue duringany of the above procedures. As is known, reperfusion may cause aspontaneous arrhythmia to occur after cardiac surgery.

In some embodiments, PRP may be used to treat any lung disease. Examplesof lung disease include, but are not limited to: Acute RespiratoryDistress Syndrome (ARDS), Alpha-1-Antitrypsin Deficiency,Asbestos-Related Lung Diseases, Asbestosis, Asthma, Bronchiectasis,Bronchitis, Bronchopulmonary Dysplasia (BPD), Chronic Bronchitis (seeCOPD), Chronic Obstructive Pulmonary Disease (COPD), Collapsed Lung (seeAtelectasis), Cough, Cystic Fibrosis, Emphysema (see COPD), Hemothorax,Idiopathic Pulmonary Fibrosis, Infant Respiratory Distress Syndrome(Respiratory Distress Syndrome in Infants), LAM(Lymphangioleiomyomatosis), Lung Transplant, Pleural Effusion, Pleurisyand Other Pleural Disorders, Pneumonia, Pneumonoconiosis, Pneumothorax(see Pleurisy and Other Disorders of the Pleura), Pulmonary Embolism,Pulmonary Arterial Hypertension, Pulmonary Fibrosis (see IdiopathicPulmonary Fibrosis), Respiratory Distress Syndrome in Infants,Respiratory Failure, Sarcoidosis, Tracheostomy, andVentilator/Ventilator Support. In some embodiments, PRP compositions aredelivered directly to the lung via bronchoscopy or delivering indirectlyto the lung via the heart or blood vessel. Measurements of tissueperfusion or function may be done to evaluate the efficacy of thetreatment.

In some embodiments PRP is useful in treatment of disease and conditionsin a variety of tissues including but not limited to heart, lung, liver,kidney, brain, spinal cord, muscle, tendon, bone, skin, ligaments andany other body cell or tissue. Rotator Cuff Tendinitis or Tear, RotatorCuff Impingement Syndrome or Bursitis, Bicipital Tendinitis, labrumtears, arthritis, instability DeQuervaine's Tenosynovitis, arthritis,other wrist or finger tendinitis, ligament tears or dysfunction of thefingers Illiotibial Band Tendinitis (ITB Syndrome), Psoas Tendinitis andbursitis, Greater Trochanteric Bursitis, Hip labrum tears, PiriformisSyndrome, Sacroiliac Joint Dysfunction, arthritis Patellar Tendinitis,Patellar Femoral Syndrome, chondromalacia patella, partially torn orstrained major ligaments of knee (ACL/LCL/MCL), meniscus tears,arthritis, patellar instability Achilles Tendinitis, PeronealTendinitis, arthritis, recurrent ankle sprains, other foot or ankletendinitis Whiplash injuries, headaches related to the neck, arthritisFacet joint arthritis, rib problems, and pain associated with scoliosis.In some embodiments, PRP compositions may be used to treat disogenicspine pain or disorders alone or in combination with other treatments.

As used herein the term “an effective amount” of an agent is the amountsufficient to treat, inhibit, or prevent ischemia and/or reperfusioninjury associated with indications and conditions including, but notlimited to, myocardial infarction, arteriosclerosis. stroke, septicshock, traumatic shock, and associated with surgical procedures such asvascular interventional procedures including angioplasty, surgery thatinvolves restriction of blood supply to an organ or tissue, abdominalsurgery, abdominoplasty, adenoidectomy, amputation, angioplasty,appendectomy, arthrodesis, arthroplasty, brain surgery, cesareansection, cholecystectomy, colon resection, colostomy, cornealtransplantation, discectomy, endarterectomy, gastrectomy, grafting ofskin or other tissues, heart transplantation, liver transplantation,heart surgery hemicorporectomy, hemorrhoidectomy, hepatectomy, herniarepair, hysterectomy, kidney transplantation, laminectomy, laryngectomy,lumpectomy, lung transplantation, mammoplasty, mastectomy,mastoidectomy, myotomy, nephrectomy, nissen fundoplication,oophorectomy, orchidectomy, orthopedic surgery, parathyroidectomy,penectomy, phalloplasty, pneumonectomy, prostatectomy, radiosurgery,rotationplasty, splenectomy, stapedectomy, thoracotomy, thrombectomy,thymectomy, thyroidectomy, tonsillectomy, ulnar collateral ligamentreconstruction, vaginectomy, vasectomy and any surgery involving cardiacbypass, cardiac artery bypass graft surgery and organ transplantation.

In addition to the foregoing uses for the compositions, methods andsystems described herein, it will be apparent to those skilled in theart that other injured tissues, in addition to injured cardiac tissueand connective tissue, would benefit from the delivery of structuralsupport materials to treat the injuries. Non-limiting examples of suchtissues include the stomach, to reduce food intake and increase satiety;the abdominal wall, to prevent and treat hernias; and the bladder toprevent or treat incontinence. Such tissues may additionally includevascular tissues.

In some embodiments, a specific cytokine, growth factor or drug isadministered to the patient to mobilize cells, PRP is prepared from thepatient's blood and is used to treat cartilage injuries or disorderssuch as knee osteoarthritis or spinal disc degeneration.

In some embodiments, a mobilization agent is provided subcutaneously,intravenously or intramuscularly for 1-5 days to the patient, PRP isprepared from the patient and injected intramuscularly to treat theheart or into the muscle of the leg to treat peripheral vasculardisease.

In some embodiments, a mobilization agent is administered to the patientand the PRP is prepared. This PRP is then used in an activated orunactivated manner to treat the wound.

It is noted that the PRP may be obtained from the person to be treated(autologous) or the PRP may be obtained from one individual butadministered to a different individual.

PRP prepared as described by any of the above methods may be used toprovide the platelet concentrations in the following plateletformulations. Further, in some embodiments, the compositions andcomponents for created and utilizing the compositions as disclosedherein can be assembled into kits to be used by laboratory technician,doctor, medical professional, or others assembling such compositions foruse.

Platelet Formulations Example One

Platelets Concentration: 151,000/microliter to 7,000,000/microliter orhigher.

CD34+ Cell Concentration: 1-3×10⁹ per liter to 100×10⁹ per liter orhigher.

Platelet and CD34+ cell concentrations may be at any level in betweenthe above values. The PRP could be unactivated or activated.

Example Two

Platelets Concentration: 151,000/microliter to 7,000,000/microliter orhigher.

CD34+ Cell Concentration: 1-3×10⁹ per liter to 100×10⁹ per liter orhigher.

Platelet and CD34+ cell concentrations may be at any level in betweenthe above values. The PRP could be unactivated or activated.

The composition of example one that includes:

Neutrophils, Lymphocytes, Monocytes, Eosinophils or Basophils in anyconcentration.

Example Three

Platelet Concentration: 151,000/microliter to 7,000,000 or higher.

Stem cells of any type in any concentration including embryonic stemcells, induced pluripotent stem cells, adult stem cells, and progenitorcell lines of any type.

Example Four

Platelet Concentration: 151,000/microliter to 7,000,000 or higher.

CD34+ Cell Concentration: 1-3×10⁹ per liter to 100×10⁹ per liter orhigher.

Lymphocytes, Monocytes Eosinophils or Basophils in any concentration

without any neutrophils.

Example Five

The composition of example three or four where the neutrophilconcentration is less than 100/microliter or less than 500/microliter,or less than 1000/microliter, or less than 5000/microliter or less than10,000/microliter, or less than 20,000, or less than 50,000/microliteror less than 100,000/microliter.

Example Six

Platelet-rich plasma derived from induced pluripotent stem cells.

Platelet concentration: 151,000/microliter to 7,000,000 or higher.

Pluripotent stem cells in any concentration.

Example Seven

The composition of example six within of any of the compositions ofexamples 1-6.

Example Eight

The compositions of any of examples above where the hemaglobinconcentration is less than 10 g per deciliter; preferably less than 5 gper deciliter, more preferably less than 1 g per deciliter, and mostpreferably where the hemoglobin concentration is zero.

Example Nine

The composition of any of above examples that include or do not includeother stem cells that can be derived from whole blood, bone marrow,adipose tissue or other sources.

Example Ten

The composition of any of the above examples with various concentrationsof other white blood cells including monocytes in a concentration of 0,100-500, 500-1000, 1000-5000, 5000-10, 000, 10,0000-50,000 permicroliter or higher.

Example Eleven

The composition of any of the above examples with various concentrationsof other white blood cells including lymphocytes in a concentration of 0100-500, 500-1000, 1000-5000, 5000-10, 000, 10,0000-50,000 permicroliter or higher.

Example Twelve

The composition of any of the above examples with various concentrationsof other white blood cells including neutrophils in a concentration of 0100-500, 500-1000, 1000-5000, 5000-10, 000, 10,0000-50,000 permicroliter or higher.

Example Thirteen

The composition of any of the above examples with various concentrationsof other white blood cells including eosinophils in a concentration of 0100-500, 500-1000, 1000-5000, 5000-10, 000, 10,0000-50,000 permicroliter or higher. Example Fourteen

The composition of any of the above examples with various concentrationsof other white blood cells including basophils in a concentration of 0100-500, 500-1000, 1000-5000, 5000-10, 000, 10,0000-50,000 permicroliter or higher.

Example Fifteen

A composition that contains platelets in a concentration of151,000/microliter to 7,000,000/microliter or higher that is also shownto contain stromal-derived factor −1 (SDF-1) in a concentration of 500pg/ml to 5000 pg/ml, or 100-500 pg/ml or greater than 5000 pg/ml.

Methods of Using Platelet Formulations

The platelet formulations disclosed herein may be useful in treatingdamaged connective tissue, cardiac tissue, and/or lung tissue in thesame manner as described above for PRP. Platelet formulations may alsobe useful in treating tissues with compromised blood flow, such asischemic tissue in the legs, arms, brain or other organs. Specifically,acute or chronic limb ischemia may be treated with plateletformulations. In some embodiments, the platelet formulations describedherein may repair tissue damage by slowing or halting apoptosis, andthat the anti-apoptotic effects of the platelet formulations may bemeasured based on a decrease in caspases in the blood, such ascaspase-3. In some embodiments, the platelet formulations may be appliedin conjunction with reperfusion therapy.

In some embodiments, the platelet formulations disclosed herein may beuseful in treating ischemia, cancer, a disease of the immune system, aconnective tissue injury, a skin disease, or a disease of the nervoussystem. The platelet formulations may be useful for the treatment ofacute or chronic skin conditions such as burns or wrinkles. The ischemiamay be a brain ischemia or cardiac ischemia. The cancer may be braincancer, thyroid cancer, pancreatic cancer, liver cancer, breast cancer,or prostate cancer. Other types of cancer or neoplasia may also betreated with the described platelet formulations. The connective tissueinjury may be a tendinosis, such as tennis elbow, rotator cuff injury, aknee injury, a spinal injury or plantar fasciitis. The nervous systemdisease may be Parkinsons' disease or other neurodegenerative disorderssuch as Alzheimers or Multiple Sclerosis.

Method Example 1 Use of PRP to Obtain and/or Differentiate PluripotentCell Lines

A skin biopsy (or any other appropriate tissue) is taken from anindividual and then his or her fibroblasts (or other cells) arecultured. PRP is then prepared from the whole blood of that individualor another individual and specific bioactive molecules such as vascularendothelial growth factor or a bone morphogenetic protein (or otherappropriate cytokines, chemokines or growth factors are isolated). Thesemolecules are then administered via a variety of methods to thefibroblasts and nuclear reprogramming to a pluripotent cell line isachieved.

Alternatively, an established pluripotent or multipotent cell line istreated with PRP with or without specific fractionation to inducedifferentiation of that cell line. Additional exogenous factors ormethods may be added as needed to accelerate or complete thedifferentiation of that cell line. Different preparations orcompositions of PRP may also be used to accomplish the task. In someembodiments, the preparations and compositions of PRP can be similar tobut not limited to those as described herein. PRP may further be usedalone or in combination with bone marrow concentrate, or fractions ofadipose tissue. In some embodiments, some fractions of adipose tissuecan include adipose tissue that is a source of mesenchymal stem cellsand endothelial progenitor cell or other adipose derived stem cells aswell as other cell components including preadipocytes, T cells, B cells,mast cells, adipose tissue macrophages, and other cells known in the artto be in fractions of adipose tissue. In some embodiments, the cell lineor stem cell line can be treated with a composition containing plateletsat a concentration of 151,000/microliter to 7,000,000/microliter orhigher in combination with bone marrow concentrate and/or any fractionof adipose tissue.

In some embodiments, the composition can include progenitor or precursorcells combined with the PRP composition to aid in acceleration orcompletion of the differentiation of a cell line. Progenitor orprecursor cells in tissue are partially differentiated, usuallyunipotent cells, which give rise to the differentiated cells. Theprogenitor or precursor cells may have lost most or all of the stem cellmultipotency. Progenitor or precursor cells can be combined with the PRPcomposition to accelerate or complete the differentiation of a cellline. Therefore, in some embodiments, the cell line or stem cell lineculture can include a composition containing platelets concentration of151,000/microliter to 7,000,000 or higher in combination progenitor orprecursor stem cell lines of any type including mesenchymal stemprecursor lines.

In some embodiments, the cell line or stem cell line culture can includea composition containing platelets or PRP in combination with embryonicstem cells, induced pluripotent stem cells, adult stem cells (such asmesenchymal stem cells), and or progenitor cell lines of any type. Thecomposition containing platelets can have a concentration of151,000/microliter to 7,000,000 or higher.

The cells lines would be carefully monitored for differentiation bysurface markers or other measures of cell specificity. PRP could furtherbe used to accelerate proliferation of the cell line. One specificexample would be to create functional human platelets from inducedpluripotent stem cells alone or then use them to create platelet-richplasma of any composition. The iPSC would first be differentiated intomegakaryocytes via a variety of vectors. Concentrated platelets wouldthen be harvested and suspended in plasma, saline or other biologicmedia. In some embodiments, the PRP produced with this method can be acomposition of platelet rich plasma derived from induced pluripotentstem cells at a platelet concentration of 151,000 to 7,000,000 or higherand pluripotent stem cells in any concentration or form.

Further, in some embodiments, the compositions and components forcreated and utilizing the compositions as disclosed herein can beassembled into kits to be used by laboratory technician, doctor, medicalprofessional, or others assembling such compositions for use.

Method Example 2 Cell Differentiation using Platelet-rich Plasma

Glioblastoma cells (012705), a cell type with severe dedifferentiationand an anaplastic phenotype were cultured with and without 10% PRP for10 days. At 10 days, dramatic differences were noted in the phenotypicexpression of the cell lines.

Without PRP, the cells continued to be of an anaplastic phenotype. WithPRP, the cells differentiated into a mature phenotype resembling anastrocyte. See Figure.

Further, in some embodiments, the compositions and components forcreated and utilizing the compositions as disclosed herein can beassembled into kits to be used by laboratory technician, doctor, medicalprofessional, or others assembling such compositions for use.

Drug Discovery

Platelet-rich plasma (PRP) could be used to identify or validate a newdrug. Specific targets such as proteins, genes, RNA, microRNA, orepigenetic modifications or markers would be tested using whole orfractionated PRP of any type or formulation. Assays would be developedusing PRP to look for disease associations, genetic expression,phenotypic or genotypic alternations in-vitro or in-vivo markers.Specific fractions of PRP could also be developed by blocking specificgrowth factors to create more bioactive PRP. Importantly, novel factorswithin PRP could be identified, purified and either used therapeuticallyor created via genetic engineering techniques to produce a drug. Drugscould further be tested in cell cultures that use PRP as part or all ofthe growth media. Toxicity or potency of a drug could further bemeasured its influence on PRP. Specifically, PRP could be used in targetidentification and validation of drugs.

PRP Potency

PRP potency could be measured visually by observing over time thechemotaxis of stem cells or other bioactive molecules. PRP potency couldalso be simply measured by visually quantifying platelet interactionwith other cellular components or via the activity of platelets to aknown agonist such as calcium, collagen or thrombin. This could be donevia single observations under a microscope or via time lapse photographyto measure cellular movement or chemotaxis. Time or completeness ofplatelet activation could be another endpoint. For example, in someembodiments, the potency of platelet rich plasma can be visuallyobserved over time by any method known in the art or described hereinincluding but not limited to cellular movement, microfluidics orchemotaxis. In some embodiments, the measurement of PRP potency can beobserved via visual means such as a microscope or its activity inrelation to other cells

What is claimed is:
 1. A method of producing induced pluripotent stem(iPS) cells comprising: obtaining fibroblast cells from skin tissue froman individual, administering a growth factor selected from the groupconsisting of vascular endothelial growth factor (VEGF), granulocytecolony stimulating factor (G-CSF), plerixafor, sargramostim,γ-tocotrienol, vitamin E, ancestim and bone morphogenic growth factor tothe individual, isolating platelet-rich plasma (PRP) from whole blood ofthe individual, and treating the fibroblast cells of the individual withthe isolated PRP.
 2. The method of claim 1, wherein the whole blood isobtained from spleen or bone marrow.
 3. The method of claim 1, whereinthe platelet-rich plasma comprises a platelet concentration of151,000/microliter to 7,000,000/microliter or higher, and furthercomprising treating the fibroblast cells of the individual with a bonemarrow concentrate and fraction of adipose tissue.
 4. The method ofclaim 1, wherein the platelet-rich plasma comprises a plateletconcentration of 151,000/microliter to 7,000,000/microliter or higher,and further comprising CD34+ cells at a concentration 1-3×10⁹ per literto 100×10⁹ per liter or higher.
 5. The method of claim 1, wherein theplatelet-rich plasma comprises a platelet concentration of151,000/microliter to 7,000,000/microliter or higher, and furthercomprising stromal-derived factor −1 (SDF-1) in a concentration of 100pg/ml to 5000 pg/ml.
 6. The method of claim 1, further comprisingmeasuring the potency of platelet rich plasma by visually observing itover time by at least one method selected from the group consisting ofcellular movement, microfluidics, and chemotaxis.
 7. The method of claim1, wherein the PRP is used in drug discovery to identify new targets. 8.The method of claim 1, wherein the PRP is used for nuclearreprogramming, cell proliferation, or differentiation.
 9. The method ofclaim 1, further comprising administrating granulocytecolony-stimulating factor (G-CSF) or any other molecule that stimulatesthe production of stem cells by the body prior to the preparation ofplatelet-rich plasma from any source.
 10. A composition comprising:cells from an individual, and platelet-rich plasma (PRP) isolated fromwhole blood of the individual, wherein the cells of the individual aretreated with the PRP to produce induced pluripotent stem (iPS) cells.11. The composition of claim 10, wherein the cells are fibroblast cellsfrom skin tissue from the individual.
 12. The composition of claim 10,further comprising a growth factor selected from the group consisting ofvascular endothelial growth factor (VEGF), granulocyte colonystimulating factor (G-CSF), plerixafor, sargramostim, γ-tocotrienol,vitamin E, ancestim and bone morphogenic growth factor to theindividual.
 13. The composition of claim 10, wherein the whole blood isobtained from spleen or bone marrow.
 14. The composition of claim 10,wherein the composition comprises the platelet-rich plasma comprising aplatelets concentration of 151,000/microliter to 7,000,000/microliter orhigher and comprises at least one cell selected from the groupconsisting of embryonic stem cells, induced pluripotent stem cells,adult stem cells, and progenitor cell lines.
 15. The composition ofclaim 14, wherein the adult stem cells comprise mesenchymal stem cells.16. The composition of claim 10, wherein the composition comprises theplatelet-rich plasma comprising a platelet concentration of151,000/microliter to 7,000,000/microliter or higher and comprisesprogenitor or precursor stem cell lines.
 17. The composition of claim16, wherein the progenitor or precursor stem cell lines comprisemesenchymal stem precursor lines.
 18. The composition of claim 10,wherein the composition comprises the platelet-rich plasma comprising aplatelet concentration of 151,000/microliter to 7,000,000/microliter orhigher and comprises CD34+ cells at a concentration 1-3×10⁹ per liter to100×10⁹ per liter or higher.
 19. The composition of claim 15, whereinthe composition comprises the platelet-rich plasma comprising aplatelets in a concentration of 151,000/microliter to7,000,000/microliter or higher and comprises stromal-derived factor −1(SDF-1) in a concentration of 100 pg/ml to 5000 pg/ml.
 20. Thecomposition of platelet rich plasma comprising the platelet rich plasmaderived from induced pluripotent stem cells at a platelet concentrationof 151,000/microliter to 7,000,000/microliter or higher and pluripotentstem cells in any concentration or form.